A novel orphan receptor specific for a subset of thyroid hormone-responsive elements and its interaction with the retinoid/thyroid hormone receptor subfamily.

The steroid/hormone nuclear receptor superfamily comprises several subfamilies of receptors that interact with overlapping DNA sequences and/or related ligands. The thyroid/retinoid hormone receptor subfamily has recently attracted much interest because of the complex network of its receptor interactions. The retinoid X receptors (RXRs), for instance, play a very central role in this subfamily, forming heterodimers with several receptors. Here we describe a novel member of this subfamily that interacts with RXR. Using a v-erbA probe, we obtained a cDNA which encodes a novel 445-amino-acid protein, RLD-1, that contains the characteristic domains of nuclear receptors. Northern (RNA) blot analysis showed that in mature rats, the receptor is highly expressed in spleen, pituitary, lung, liver, and fat. In addition, weaker expression is observed in several other tissues. Amino acid sequence alignment and DNA-binding data revealed that the DNA-binding domain of the new receptor is related to that of the thyroid/retinoid subgroup of nuclear receptors. RLD-1 preferentially binds as a heterodimer with RXR to a direct repeat of the half-site sequence 5'-G/AGGTCA-3', separated by four nucleotides (DR-4). Surprisingly, this binding is dependent to a high degree on the nature of the spacing nucleotides. None of the known nuclear receptor ligands activated RLD-1. In contrast, a DR-4-dependent constitutive transcriptional activation of a chloramphenicol acetyltransferase reporter gene by the RLD-1/RXR alpha heterodimer was observed. Our data suggest a highly specific role for this novel receptor within the network of gene regulation by the thyroid/retinoid receptor subfamily.

Autocrine tumor cell growth-inhibiting activities from human malignant melanoma.

Autocrine-secreted tumor cell growth-inhibiting activities were isolated from supernatants of a malignant melanoma cell line, HTZ 19-dM, established from a central nervous system melanoma metastasis. HTZ 19-dM was characterized by cyto- and immunocytochemistry and karyotyping; cells were propagated in defined serum-free tissue culture medium for up to 8 months. Supernatants were ultrafiltrated, dialyzed, lyophilized, and purified by Bio-Gel P-10 gel permeation chromatography, leading to three active fraction pools, MIAI [melanoma-inhibiting activity (MIA), 2 kDa), MIAII (Mr 11,500-17,000) and MIAIII (proteins at the cutoff of Bio-Gel P-10) inhibiting growth of 19-dM cells with 50% inhibitory concentrations of 0.79 microgram/ml (MIAI), 0.13 microgram/ml (MIAII), and 16.7 micrograms/ml (MIAII). MIAII could be further purified by reverse phase high pressure liquid chromatography; the main activity displayed a 50% inhibitory concentration of 0.33 microgram/ml. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis one major band (molecular weight about 14,000) and two minor bands (up to Mr 17,000) were identified. Macromolecular synthesis was inhibited in 19-dM cells up to greater than 99.5%; tumor stem cell colony formation was reduced by 99.89%; the inhibitory effect of MIAII was irreversible, nonsaturable, and partially antagonized by a serum factor (depending on purification stage). MIAII was heat stable (3 min at 100 degrees C) and trypsin labile. The effect of MIAII on allogeneic neuroectodermal tumors was also investigated; proliferation of two of three malignant melanomas and two of four glioblastomas was inhibited up to 85.2%; proliferation of a neuroblastoma cell line could be inhibited to 33.8%, whereas normal fibroblasts and low grade gliomas were not influenced in their proliferation.

Melanoma-inhibiting activity inhibits cell proliferation by prolongation of the S-phase and arrest of cells in the G2 compartment.

Autocrine-secreted melanoma tumor growth-inhibiting activity (MIA, approximately Mr 8000) was isolated from supernatants of a malignant melanoma cell line HTZ-19 dM, established from a central nervous system-melanoma metastasis. Cell cycle kinetic analysis performed with bromodeoxyuridine/Hoechst flow cytometry revealed a MIA-sensitive period at the G0/G1 to S traverse; MIA mediated prolongation of the S-phase and increased arrest of cells in the G2 compartment. Growth inhibition by MIA is cell-density dependent; maximal effect is seen at low densities, and the effect may be partially antagonized by whole serum. MIA may cause growth stimulation at high cell densities and low MIA concentrations. The effect of MIA on different histological neuroectodermal cell types was compared by the same methodology: proliferation of a second malignant melanoma was inhibited, and no effect was observed with an ependymoma; 2 glioblastomas were slightly stimulated. Effects on human fibroblast-like cell strains were inconsistent. The mechanism of MIA is discussed in relation to other endogenous autocrine growth inhibitors.

Cloning of a novel malignant melanoma-derived growth-regulatory protein, MIA.

Growth and progression of malignant melanoma cells is influenced by a complex network of growth-stimulating and -inhibiting factors produced by both the tumor cells and the local environment. Here we report the purification and molecular cloning of a novel growth regulating protein, designated melanoma inhibitory activity (MIA) and provide a preliminary functional characterization. MIA is translated as a 131-amino acid precursor and processed into a mature 107-amino acid protein after cleavage of a putative secretion signal. A murine complementary DNA was isolated that encoded a MIA-protein with 88% amino acid identity. MIA is secreted into the culture supernatant by several malignant melanoma cell lines as an M(r) 11,000 autocrine growth factor and acts as a potent tumor cell growth inhibitor for malignant melanoma cells and some other neuroectodermal tumors, including gliomas. MIA has no homology to any other known protein and, therefore, represents a novel type of growth-regulatory factor. Furthermore, we describe a molecular approach to express functionally active MIA in Escherichia coli, which might be attractive as a future antitumor therapeutical substance.

In vitro studies on drug interaction of ifosfamide and ACNU in primary and metastatic human brain tumours.

Combination chemotherapy is widely employed in clinical oncology; however, there is no generally accepted model to evaluate individual tumour susceptibility to a given drug combination protocol. We therefore investigated the drug interaction of ifosfamide (4-hydroxyperoxy-ifosfamide) and ACNU in a recently developed in vitro model of paired sequential combination chemotherapy in primary and metastatic malignant brain tumours. A long-term standard [6,3-3H]-thymidine-incorporation assay, employing a liquid scintillation counting protocol, was selected to assess the drug sensitivity of human tumours. In vitro drug exposures were derived from correlating in vivo-(systemic and CNS) and in vitro-pharmacokinetic drug parameters. In combination experiments tumour cells were treated sequentially by two drugs in both sequences: drug exposures were calculated for 2 h with a 1-h drug-free interval in between. "Cut-off" concentrations (maximum in vitro exposure doses) were calculated as 1.74 microM (for primary CNS tumours: 0.58 microM) for ifosfamide and 5.4 microM (for primary CNS tumours: 1.33 microM) for ACNU. Dose/response relations were derived from isotope incorporation rates after cells had grown for approximately five population doubling times. Combination isoboles were plotted after drug doses had been transformed into "equieffective doses", enabling comparison of drug combination effects. In all three glioblastomas (with CNS exposure dose) an additive or supra-additive effect could be observed in either sequence (in one tumour a biphasic additive isobole was found for both sequences). Out of three bronchial carcinomas (small-cell type, brain metastases) in two non-identical sequences a supra-additive effect was observed in two tumours, with antagonistic effects in the third tumour. In all three malignant melanomas and in one renal carcinoma antagonistic effects were observed, whereas in a second renal carcinoma supra-additive effects were demonstrated for both sequences. We conclude that drug combination chemotherapy effects at the cellular level may be extremely heterogeneous.

In vitro studies on interaction of 4-hydroperoxyifosfamide and 2-mercaptoethanesulphonate in malignant gliomas.

Drug interference of ifosfamide and sodium 2-mercaptoethanesulphonate (MESNA) was studied in three malignant glioma cell cultures (HTZ-17, HTZ-209B, and HTZ-243) by a recently developed in vitro method for evaluation of multimodal treatment interactions. Glioma cell cultures were treated in monolayer 96-well tissue-culture plates for 2 h each, with 4-hydroperoxyifosfamide and MESNA combined in both sequences, or alone. Concentrations ranged from 0.01 microM to 50 microM in single-modality exposures, and from 0.01 microM to 10 microM in combination exposures. After five population doubling times, DNA synthesis was determined by a standard [3H]Tdr-incorporation liquid-scintillation-counting protocol. Data points were evaluated for mono- and combined treatment dose effects (adapted with a probit function), and a model-free three-dimensional response surface was created that was compared to the theoretical additive, anticipated response surface. Local additivity was analysed for any ratio of combined treatment. No tumour effects were seen with MESNA in single-drug exposure, whereas ifosfamide resulted in more than 90% inhibition of tumour DNA synthesis. In combination experiments, MESNA could be confirmed to be inert: the anticipated theoretical combination response surfaces formed a three-dimensional extension of the single-drug ifosfamide dose/response curves--the experimental combination response surfaces displayed an identical appearance (P < or = 0.05). In conclusion, these results indicate no drug interference of MESNA and ifosfamide in malignant glioma cells.

Transient thermal and mechanical response of water subject to ionizing radiation.

The ultrafast transient (10(-14) to 10(-12)S) thermal and mechanical response of water subject to ionizing radiations of different linear energy transfers has been investigated in order to understand the initial events which lead to cell mutation and lethality. Based on computational fluid dynamics, the production of a "thermal spike" around the trajectory of a charged particle and subsequent diffusion of deposited heart are calculated for particles with linear energy transfer (LET) of 4, 40, and 400 keV/microns. A radiation damage region (that is, the so-called "thermal core") is identified, and the transient behavior of the thermal core is studied. The local and transient environment has a dimension of nanometers, a scale which is of critical interest in understanding mechanisms of radiation damage in cells. The radius of the thermal core, Dd, at temperatures (or internal energy density) of up to 1,000 K, is observed to increase with LET, L, as Dd (in nanometers) = C4.L (in keV/microns)0.6, where, for example, C4 = 0.50 for T = 800 degrees C.

The effect of transforming growth factor-beta 2-specific phosphorothioate-anti-sense oligodeoxynucleotides in reversing cellular immunosuppression in malignant glioma.

This in vitro study was aimed at restitution of transforming growth factor (TGF)-beta 2-mediated suppression of T-lymphocyte activation within malignant gliomas. In early-passage tumor cell cultures of two glioblastomas (HTZ-153 and HTZ-209) and one malignant astrocytoma classified as World Health Organization Grade III (HTZ-243), autologous peripheral blood mononuclear cells were activated by interleukin-1 alpha and interleukin-2 in vitro (lymphokine-activated killer cells) and tested for cytotoxic and proliferative activity. In expression studies (Western blot and Northern hybridization) of all three tumors, TGF-beta could be detected at the protein and messenger ribonucleic acid (mRNA) levels. A polyclonal anti-TGF-beta neutralizing antibody did not enhance lymphocyte proliferation upon stimulation with tumor targets (3H-thymidine incorporation) and slightly stimulated lymphocyte cytotoxicity against autologous target cells. Preincubation of target cells for 12 hours with TGF-beta 2-specific phosphorothioate-anti-sense oligodeoxynucleotides (S-ODN's) did, however, enhance lymphocyte proliferation up to 2.5-fold and autologous tumor cytotoxicity up to 60%, compared to controls not treated with S-ODN's. Incubation of tumor cells with TGF-beta 2-specific S-ODN's resulted in decreased TGF-beta-specific immunoreactivity in cultured glioma cells, in reduced TGF-beta 2 protein concentration (Western blot), and in a change in the expression pattern of TGF-beta 2 mRNA's. These observations may have implications for in vivo and in vitro activation of a cellular immune response against autologous malignant glioma cells.

Purification and analysis of growth regulating proteins secreted by a human melanoma cell line.

Supernatants of a human malignant cell line established from a CNS metastasis, contained several proteins with putative growth regulating functions. BioGel P-10 gel filtration chromatography, reverse phase HPLC purification, and amino-terminal sequencing of purified peptides resulted in characterization of beta 2-microglobulin (beta 2M, 10 kD), ubiquitin (6 kD), and tissue inhibitor of metalloproteinases 2 (TIMP-2, 21 kD). In addition, CNBr cleavage and purification of resulting peptides revealed diazepam binding inhibitor (DBI, 8 kD) and melanoma inhibiting activity (MIA, 11 kD). The secretion of beta 2M as part of the HLA-class I complex may be related to impaired autologous anti-tumour immune function; ubiquitin may play a role in activation or deactivation of extracellular proteins or cell-cell interactions. As HTZ-19 cells respond in a dose-dependent manner to midozolam, DBI may interfere with growth regulation mediated by diazepam receptor sites. In a collagenolytic assay, TIMP-2 interfered with metalloproteinase functions, which are required for degradation of collagen type IV and organotopic metastasis. MIA is clearly associated with a proliferation inhibiting effect on HTZ-19 cells. In conclusion, although this tumour shows a degree of progression, several proteins with putative functions at different cellular levels were identified, related to proliferation as well as to the type of metastasis.

In vitro studies on interaction of 4-hydroperoxy-ifosfamide and radiotherapy in malignant gliomas.

Drug-radiation multimodal chemotherapy for malignant brain tumor cells was stimulated in vitro by a recently developed computer program, based on evaluation of 3D-response surfaces and interaction isoboles. Three malignant glioma tumor cell cultures (HTZ17, 146 and 209 B) were sequentially treated in vitro by increasing doses of 4-hydroperoxy-ifosfamide, according to in vivo/in vitro pharmacokinetic correlation (0.01 to 10 microM), and increasing single doses of Gamma-radiation (clinical60Co-radiotherapy unit, 0.26 to 4 Gy or 1 to 3.38 Gy, respectively). After approximately five population doubling times with standard tissue culture conditions, 3H-Tdr-incorporation was determined by a liquid scintillation counting protocol. Data points were evaluated for mono- and combined treatment dose-effects. A model-free 3D-response surface was created and compared to the theoretical additive response surface. Local additivity was analysed for any desired ratio of combined treatment as well as for isoboles. No significant sub- or supraadditive effects were observed, indicating additive effects in all 3 tumors. No sequence dependence of effects could be demonstrated. In case radiotherapy and ifosfamide-chemotherapy are active treatment modalities and additive effects are found, we conclude that the combination of ifosfamide and radiotherapy might be attractive for the treatment of malignant brain tumors and should be further studied.

MIA, a novel serum marker for progression of malignant melanoma.

MIA was isolated previously as a small soluble protein secreted from malignant melanoma cell lines in vitro. Highly restricted expression patterns in melanocytic tumors were identified in vivo. We therefore quantitated serum levels of MIA protein by means of a non-radioactive ELISA and investigated whether MIA provides clinically relevant parameters in patients with malignant melanomas. Here we report enhanced MIA serum levels in 13% and 23% of patients with stage I and II disease, respectively, and in 100% with stage III or IV disease. Response to therapy in stage IV disease correlated with changes in MIA serum levels and surgical removal of metastases led to normalization of serum values. Repeated measuring of sera from 350 patients with a history of stage I or II melanoma during follow-up, we detected 32 patients developing positive MIA values. At the time of serum analysis 15 of them had developed metastases and one presented with metastatic disease 6 months later. In conclusion, MIA represents a novel serum marker for systemic malignant melanoma revealing the highest sensitivity and specificity among currently available markers.

Gauging the likelihood of cavitation from short-pulse, low-duty cycle diagnostic ultrasound.

Although no deleterious effects form diagnostic ultrasound have been reported in epidemiologic studies and surveys of widespread clinical usage (Ziskin and Petitti 1988), the conditions for the onset of transient cavitation must be investigated in the total evaluation of potential risks associated with diagnostic ultrasound applications. An extension of the results from the approximate theory developed by Holland and Apfel (1989) is applied in this paper to a population of nuclei to predict the onset of cavitation in host fluids with physical properties similar to those of biological fluids. From this analysis and from results of recent in vitro cavitation experiments, an index is developed which can gauge the likelihood of substantial microbubble growth in the presence of short-pulse, low-duty cycle diagnostic ultrasound.

Method for determining the reliable prediction(s) of compositions of tissue phantoms.

In a previous paper, we showed that mixture laws can be used to predict the compositions of tissue phantoms. Due to the variation in the performance of the mixture laws when they are applied to phantoms, however, it is difficult to determine which predictions of the composition are most reliable. In this paper, we study the causes of the variation in the performances of different sets of mixture laws, and propose a criterion for choosing a reliable predictor. The predictions selected with the proposed criterion agree very well with the known compositions of phantoms. The potential uses of the mixture methodology and the criterion to tissue characterization are discussed.

Applications of mixture laws for predicting the compositions of tissue phantoms.

This paper generalizes the mixture law methodology for ultrasonic tissue characterization by combining mixture laws used by Apfel and Seghal et al. in producing nine different combinations. After applying these combinations of mixture laws to 10 tissue phantoms, it is shown that three of the combinations consistently give better predictions of the composition of the phantoms than others. The results verify directly that the phase shift parameter law is probably not a good choice in predicting tissue composition. The potential uses of the mixture methodology are discussed.

In vitro measurements of inertial cavitation thresholds in human blood.

Inertial cavitation thresholds were measured in human blood exposed to pulsed ultrasound. Freshly drawn blood, bank blood and aqueous dilutions of both were used in this experimental study. Micrometer-sized polystyrene particles were used as extra potential nuclei in some samples. Focused transducers with megahertz center frequencies (2.5 MHz, 4.3 MHz) were employed to generate pulsed ultrasound to induce cavitation. Specially designed cells for hosting the blood samples were made to adapt to the experimental environment. Cavitation threshold measurements were achieved by using an active cavitation detection scheme which utilizes a highly focused transducer with a much higher center frequency (30 MHz). In 50% diluted blood samples, when no polystyrene particles were added to the samples, the threshold for cavitation was about 4.1 MPa at 2.5 MHz, while no cavitation was detected at 4.3 MHz. Generally, the measured thresholds decrease in samples with lower volume concentration of red blood cells or when polystyrene particles were added to the samples. Results show that the measured thresholds in some circumstances are in the range of output pressure of diagnostic ultrasound instrumentation; but for whole, freshly drawn blood, our apparatus was unable to detect cavitation, even at 6.3 MPa.

Direct evidence of cavitation in vivo from diagnostic ultrasound.

Recent increases in the pressure output of diagnostic ultrasound scanners have led to an interest in establishing thresholds for bioeffects in many organs including the lungs of mammals. Damage may be mediated by inertial cavitation, yet there have been no such direct observations in vivo. To explore the hypothesis of cavitation-based bioeffects from diagnostic ultrasound, research has been performed on the thresholds of damage in rat lungs exposed to 4.0-MHz pulsed Doppler and color Doppler ultrasound. A 30-MHz active cavitation detection scheme complementing these studies provides the first direct evidence of cavitation in vivo from diagnostic ultrasound pulses.

Effects of diethylstilbestrol (DES) medication during pregnancy: report from a symposium at the 10th international congress of ISPOG.

This article is a report based on presentations at the symposium Effects of Diethylstilbestrol (DES) Medication during Pregnancy at the conference Reproductive Life, 10th International Congress of The International Society of Psychosomatic Obstetrics and Gynaecology (ISPOG) in Stockholm, Sweden June 14-17, 1992. The objective of this symposium, chaired by Eylard van Hall and Ingar Palmlund, was to provide the basis for a discussion of how the risks of DES had been evaluated in different countries. In the general discussion following the presentations everybody present expressed concerns that DES might still be given to pregnant women in many parts of the world, called for measures to alert medical professionals world-wide to the hazards of the use of DES, and requested measures to effect an international ban of DES in medication for pregnant women.

Versatile resonance-tracking circuit for acoustic levitation experiments.

Objects can be levitated by radiation pressure forces in an acoustic standing wave. In many circumstances it is important that the standing wave frequency remain locked on an acoustic resonance despite small changes in the resonance frequency. A self-locking oscillator circuit is described which tracks the resonance frequency by sensing the magnitude of the transducer current. The tracking principle could be applied to other resonant systems.

Observations on the psychological impact of diethylstilbestrol exposure and suggestions on management.

The emotional impact of diethylstilbestrol (DES) exposure is described in a series of 50 mothers and daughters interviewed by psychiatrists. Patterns of response to this trauma and methods of resolution are discussed, and opportunities for preventive intervention by gynecologists are suggested. Specific, open dialogue about DES with the patient as a colleage can minimize the emotional sequelae of the experience.

PIP: This study analyzes the emotional impact of diethylstilbestrol (DES) exposure in an index population consisting of 50 women at risk plus 30 mothers who were all interviewed about their DES experience in an open-ended, in-depth, clinical style. The findings show that significant emotional upset is the normal response to the knowledge that the ingestion of a drug during pregnancy can cause or has caused some abnormality in the offspring. Nevertheless, the capacity of a woman to come to terms with the anxiety DES has generated, once she had been given the chance to express her feelings and fears, was impressive. DES daughters reacted to the DES experience in one of 3 ways, in descending order of frequency: 1) trust (80%). Most DES daughters rationalized that their mothers and doctors did the best they could, and were generally cooperative in their follow-up care; 2) hostility (10%); and 3) fear (10%). 90% of DES mothers came to terms with the knowledge and implications of DES exposure in ways characteristic of their life-long personality styles; in contrast, the remaining 10% who did not come to terms with the reality of DES exposure felt overwhelmed by quilt, paranoid rage, fear, and despair. Physicians can help patients deal with such problems by: 1) acknowledging problematical feelings and expecting them to be difficult to deal with; 2) noting the patient's pattern of response, and supporting her strengths; 3) giving factural information matter-of-factly; 4) listening to reactions to this information; 5) giving a structured plan in which the woman participates and be available for follow-through on it (eg, periodic colposcopic examinations); and 6) referring the women to support groups for an extended network of information and continued support.

An improved theory for the prediction of microcavitation thresholds.

An approximate analytical formulation is presented that allows for the calculation of acoustic pressure thresholds for transient cavitation over a variety of frequencies and host fluid parameters. Specifically, R.E. Apfel's (1986) theory is extended to include an estimate of the time delay associated with the Laplace pressure, 2sigma/R(0), where sigma is the surface tension and R(0) is the initial radius. Also presented is a correction factor for the time-averaged pressure difference, across the bubble wall during growth. An optimum size distribution of nuclei for the predisposition of a sample to microcavitation is exhibited. The role of transient cavitation in medical ultrasound is discussed.